MicroRNAs (miRNAs) are ~22 nt long, single-stranded RNA guides that regulate the expression of plant and animal genes. miRNAs regulate more than one-third of all human genes, and inappropriate production or loss of microRNAs in specific types of cells can cause human disease. Thus, a better understanding of how miRNAs are made and how they function will advance knowledge of the causes of and potential treatments for human diseases. The goal of these studies is to understand how the information in these tiny riboregulators and their precursors is used by higher eukaryotes to ensure the accurate production of mature miRNAs, their loading into functional protein complexes, and the appropriate type of regulation of authentic mRNA targets. miRNAs were discovered in 1993 and the study of their biogenesis began in 2001 with the discovery that the multi-domain ribonuclease III (RNase III) enzyme Dicer converts miRNA precursors (pre-miRNAs) into mature miRNAs. In the intervening years, the number of known and predicted miRNAs has grown from two to more than 10,000. As a class, miRNAs may rival transcription factors in their importance for orchestrating changes in gene expression. This proposal uses Drosophila melanogaster to study the biogenesis and assembly of miRNAs into functional complexes, as well as the regulation of mRNA targets by miRNAs, because these processes are closely conserved between flies and humans. A combination of cell-free biochemical methods, cell culture experiments, cell biology, and in vivo genetics in flies will be used to dissect the mechanism and biological importance of the miRNA pathway. What is learned in flies is tested in mammalian cell extracts, and in vitro in cultured human and mouse cell lines to identify where these processes are conserved and where they diverge between flies and mammals. The next grant period will continue efforts to understand miRNA biogenesis and function. The project seeks to identify the proteins and protein complexes required to produce miRNA from pre-miRNA and to determine how these proteins function in miRNA maturation; determine how small RNAs are sorted into complexes containing different Argonaute proteins; determine how miRNA stability is controlled; and identify the enzymes that tail and trim Ago1-bound miRNAs.